This is a revised application for continued support to study mitochondrial contributions to Ca2+ regulation in wild type and mutant SOD1G93A mouse motor nerve terminals. Changes in cytosolic and intramitochondrial [Ca2+] evoked by repetitive stimulation will be measured by imaging the fluorescence of indicator dyes. Phasic (end-plate potentials) and asynchronous quantal transmitter release will be recorded electrophysiologically, and vesicular recycling measured using styryl dyes. We have shown that mitochondrial uptake of Ca2+ is critically important for sustaining neuromuscular transmission during high-frequency stimulation. Proposed experiments will investigate the mechanisms by which motor terminal mitochondria take up, store and extrude this Ca2+ load. Using a novel permeabilized motor terminal preparation we will test how certain cytosolic components affect the affinity of mitochondrial Ca2+ uptake. We will test the role of inorganic phosphate in intramitochondrial Ca+ buffering, and investigate the linkage between mitochondrial Ca2+ extrusion and cytosolic [Na+]. The impact of Ca2+ uptake on mitochondrial energy metabolism will be investigated using measurements of stimulation-induced changes in mitochondrial membrane potential and changes in NADH and FAD autofluorescence. Another series of experiments will investigate the linkage between abnormal mitochondrial [Ca2+] handling and abnormal transmitter release in a mouse model of familial amyotrophic lateral sclerosis (ALS, SOD1G93A). Embryonic SOD1G93A motoneurons are especially vulnerable to nitric oxide (NO)-induced death. NO is produced in active muscle, so we will investigate whether the function of SOD1G93A motor terminals is more susceptible to NO-induced disruption than that of wild-type terminals, to test the hypothesis that NO contributes to functional deficits and degeneration of motor terminals in these mice. The proposed studies will thus probe fundamental mechanisms concerning how motor terminal mitochondria handle physiological Ca2+ loads, and investigate how Ca2+ dysregulation and NO contribute to motor terminal dysfunction in SOD1G93A motor terminals.